A classic example of a sequential chemical reaction is the Edman protein sequencing technique involving the stepwise removal and identification of the N-terminal amino acid residues of a protein. The traditional Edman technique involves coupling the N-terminal amino acid residue to phenylisothiocyanate (PITC) in a solvent under alkaline or anhydrous conditions to form a phenylthiocarbamyl derivative. Excess PITC (usually at least 100 fold molar excess) is removed, typically by liquid extraction and the solvent also removed. The N-terminal amino acid, coupled to the PITC is subjected to cleavage by anhydrous acid to form an anilinothiazolinone (ATZ) derivative of the amino acid. The ATZ derivative is removed for subsequent chromatographic identification of the amino acid portion. The original protein is thus truncated, at its N-terminal by one amino acid allowing access to the formerly penultimate amino acid to be coupled, cleaved and identified with a subsequent Edman cycle. Further cycles can be undertaken to determine the entire sequence of the protein.
Some of the disadvantages of the traditional liquid phase Edman technique include the necessity to introduce plural incompatible reagents to achieve the coupling and cleavage steps for each cycle. In particular it is noted that strong alkali conditions are required in the coupling step whereas a strong acid environment achieves the cleavage. Clearly differentiation of these strong reagents is required to ensure a consistent removal of all the N-terminal residues without also generating spurious derivatives or uncoupling further non terminal residues. Furthermore the requirement for removal of the various volatile and non volatile auxiliary reagents and solvents during each cycle leads to sample loss and/or the formation of insoluble by-products. Relatively large volumes of sample and reagents are also required.
Effective automation of the Edman technique is first described in Edman and Begg, "A Protein Sequenator", in the European J. Biochem. 1, (1967), 80-91 and in U.S. Pat. No. 3,725,010. In these sequenators a liquid phase Edman technique is carried out in a thin film formed on the inside wall of a rotating reaction cell, now termed a "spinning cup". The spinning cup is located within a closed reaction chamber to maintain an inert atmosphere. Reagents are added to the cup by a system of pumps and valves and material removed by overflow over the lip of the spinning cup, vacuum evaporation or by dissolving or extracting in non polar solvents. The liquid reagents and solvents themselves form films on the walls of the spinning cup which pass over and interact with the sample film as the cup spins. The reagents dissolve the sample film and perform the coupling and cleavage stages of the Edman process, after which volatiles are removed by evacuation and the remaining sample film solvent extracted to transfer resulting amino acid thiazolone for identification.
It will be apparent that this dynamic system, requiring fluid and vacuum seals is difficult to construct and operate. The agitation induced by the spinning cup can cause the sample film to be overly extracted during washing or dislodged, in particular if the small protein being sequenced is a polypeptide. The initial protein sample must therefore be relatively large in volume and chain length. The drying of the protein onto the inner wall of the spinning cup must also be performed very carefully and slowly to achieve an even thin film while avoiding boiling and splatter during desiccation. Precise metering of reagents and solvents into the cup is also required to ensure consistency between cycles.
Laursen, in the European J. Biochem. 20 (1971), and Waschter et al., FEBS LeH 35, 97 (1973), described alternative automatic sequencers in which the sample is immobilized by covalent linkage to the surface of a bead matrix/gel type solid phase. This allows all reagents and solvents to be removed by solvent replacement rather than vacuum evaporation or other drying. Solvent replacement techniques, however, necessitate covalent linkage of the sample to avoid washout by the solvents used. U.S. Pat. Nos. 4,704,256, 1,610,847 and 4,603,114 relate to similar technologies in which the sample is embedded in a permeable matrix and subject to liquid solvents.
Drawbacks of the solvent systems as discussed above have led to gas solid phase Edman techniques, for instance as described in U.S. Pat. Nos. 3,892,531 and 4,065,412. In the former patent the sample is attached to a finger like extension within a reaction chamber, while the latter applies the sample to inner and outer surfaces of macroporous beads. In each system at least one of the reagents is introduced in the gaseous form; however neither system is amenable to contamination free, multiple cycles due to inefficiencies in washing by dew formation on the finger or by channelling of the solvent between rather than through the beads.
Each of the above mentioned sequenators have required undesirably large samples due to various inefficiencies in washing etc. British Application No. GB 2146550 describes an attempt to miniaturize a reaction flask suitable for Edman technique having capillary tubes to spray wash interior walls bearing an immobilizing sample matrix. Fluids within the flask can be agitated by the introduction of a gaseous phase. There is still, however, a need for systems more amenable to miniaturization and using even smaller samples. There is also a need for systems allowing direct interfacing of the chemical reactor with identification or assay apparatus such as mass spectrometers, chromatographs etc.
The above described Edman technique for terminal degradation of a protein, successively removed N-terminal residues. Alternative systems using a C-terminal degradation, such as phosphoryl mercaptobenzothiazole or benzoyl isothiocyanate are described in "Methods in Protein Sequence Analysis" (1991) Jornvall/Hoog/-Gustavsson (Eds) Birkhauser Verlag, Basel. The latter system embodies a two stage couple/cleave reaction between the cleaving reagent and C-terminal amino acid, rather like the Edman technique. Further guidance on protein sequencing is founding in Schlack et al. (1926) Hoppe-Scylers, Z. Physiol. Chemie., 154, 126-170 and Stark (1972) "Methods in Enzymology", Hirs/Timascheff (Eds) Academic Press, 25, 369.
The above description of previous techniques and apparatus has centered on protein sequencing, but it will be readily apparent that many other chemical processes face similar problems of sample loss or contamination, inefficient or overly vigorous washing, inefficient access of reagents etc. Examples of such reactions include site specific modification, construction or sequencing of other macromolecules.
In work leading up to various aspects of the present invention, the inventors also became involved in the development and application of microbore column liquid chromatographic techniques that are compatible with high-sensitivity microsequencing methodologies.
Compared to their conventional column counterparts (4.6-mm Internal Diameter (I.D.)), microbore columns (1-2.1-mm I.D.) offer enhanced mass sensitivity (5-20-fold) and decreased peak volumes (40-60 .mu.l) without any striking diminution of resolution. Microbore column liquid chromatography is now widely used for peptide mapping with proteases, complete protein structure determinations, isolation of proteins form acrylamide-gel electroeluates and detergent mixtures. More recently, microbore chromatography has been used in tandem with mass spectrometry.
Although there has long been a desire to further miniaturise liquid chromatography, progress has been restricted to the availability of packed capillary columns and instrumentation designed to facilitate the operation of such columns.